Boron-containing aptamers to ATP

Abstract

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.

Figure 1

Boranophosphate-substituted nucleotides used in this study, shown in Sp configuration.

Figure 2

The effect of including boronated nucleotides on target binding for aptamers that had originally been selected with normal NTPs to bind various nucleotide cofactor targets. y-Axis is percent RNA eluted from corresponding affinity resins when RNAs were transcribed with normal NTPs (shaded), bG in place of GTP (black) or bU in place of UTP (white). Aptamer wt204 is the 65 nt element identified through boundary analysis of an ATP-binding aptamer from the present selection. Only the last two of this set were assayed with bU. Identities of other aptamer and resins are given in the text.

Figure 3

The ζ-motif ATP aptamer. N, any nucleotide; n, any nucleotide that pairs with N. Stippled bar represents an AMP moiety intercalated between A10 and G11.

Figure 4

Sequences from selections with bG or bU substitutions or with normal nucleotides. Nucleotide numbers are given above the sequences (primer-binding sequences not shown). Functional boundaries, where measured, are given to the right of the corresponding sequences, with the form (5′-boundary)/(3′-boundary), including uncertainty in the measured boundary; nd, not determined. The signature sequence of the ζ-motif is underlined (the motif from bU321 is formed in part by the 5′-primer-binding sequence). The number of occurrences of nearly identical sequences is given in parentheses next to the sequence name. The complete sequence data set is available at http://ernie.chem.indiana.edu/dhburke/all_seqs.html.

Figure 5

Effect of including boronated nucleotides on binding to the ATP resin for aptamers selected with either (A) bU in place of UTP or (B) bG in place of GTP. Labels for y-axis and column shadings are as indicated in the legend to Figure 2.

Figure 6

Predicted structure of bG40min, a truncated version of isolate bG40 identified through deletion and 3′ functional boundary analysis, as predicted by mfold (38). Lower case letters indicate the fixed primer-binding sequence.

Figure 7

Recognition of ATP analogs by aptamer bG40. Uniformly radiolabeled aptamer was bound to ATP resin and washed with binding buffer, then eluted with 0.5 mM analog (fractions 1–6), followed by elution with 5.0 mM ATP (fractions 7–12, after arrow). Analogs used are listed on the figure in the same order, from top to bottom, as the corresponding traces.